hsp70 is not required for high affinity binding of purified calf uterine estrogen receptor to estrogen response element DNA in vitro.
Bovine estrogen receptor (ER) was purified to near homogeneity by estrogen response element (ERE) affinity chromatography, and its ERE binding ability was measured in vitro. Highly purified ER bound EREs with reduced affinity compared to partially purified ER. Partially purified ER contained hsp70, but highly purified ER did not. We examined whether addition of purified recombinant human hsp70 or purified bovine hsp70 would restore the higher ERE binding affinity, stoichiometry, and ligand retention detected with partially purified receptor and how hsp70 affected the rate of ER-ERE association and dissociation. ER-ERE binding was not affected by antibodies to either constitutive or induced forms of hsp70, regardless of ER purity. Addition of purified hsp70, with or without ATP and Mg2+, did not affect the association or dissociation rates of highly purified liganded ER binding to ERE. hsp70 Did not alter the total amount of ER-ERE complex formed. Similarly, hsp70 did not affect the rate of [3H]estradiol (E2) or [3H]4-hydroxytamoxifen (4-OHT) ligand dissociation from ER in the presence or absence of EREs. These data contrast with a report showing that maximal ERE binding by highly purified recombinant human ER required hsp70. We conclude that ER, purified from a physiological source, i.e., calf uterus, does not require hsp70 for maximal ER-ERE binding in vitro. Additionally, once ER is activated and bound by ligand, the receptor assumes its proper tertiary structure, and hsp70 does not impact ER ligand binding domain conformation.
Klinge
C M
CM
Department of Biochemistry, The University of Louisville School of Medicine, KY 40292, USA. cmklin01@ulkyvm.louisville.edu
Brolly
C L
CL
Bambara
R A
RA
Hilf
R
R
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism*; D004967:Estrogens / Q000378:metabolism*; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011960:Receptors, Estrogen / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014599:Uterus / Q000378:metabolism*
D004967:Estrogens; D018840:HSP70 Heat-Shock Proteins; D011960:Receptors, Estrogen; D004247:DNA